This renewal application proposes extensive studies focusing on the molecular genetics of the cutaneous basement membrane zone (BMZ) towards delineating the molecular basis of various forms of epidermolysis bullosa (EB). The proposed studies are designed to test the hypotheses that genetic lesions in the BMZ structural genes underlie various forms of EB, and that the precise phenotype and mode of inheritance depend on the types and combinations of specific mutations. This application is based on solid progress in this project, including: (a) cloning of the entire human type VII collagen cDNA and the corresponding gene (COL7A1); (b) elucidation of intron-exon organizations for the three laminin 5 genes, LAMA3, LAMB3, and LAMC2, as well as for BPAG2 and ITGB4, candidate genes for junctional forms of EB (JEB); (c) delineation of over 60 distinct mutations in COL7A1 in the dystrophic forms of EB; (d)identification of distinct mutations in the five candidate genes for JEB. This proposal details continuation of concentrated, multidisciplinary, and inter-institutional studies by investigators at the Jefferson Institute of Molecular Medicine and the MGH/Harvard Cutaneous Biology Research Center. The five component projects are highly interdependent. Project I, "Genetic Linkage Analysis and Positional Candidate Gene Cloning for Genodermatoses," is vital to rule in or rule out specific genes and alleles as candidate genes for mutations in families with EB. This project will also map new keratinocyte-specific expressed sequence tabs. Project 2. "Cloning and Characterization of Novel Genes Expressed in the Skin," will provide new gene probes and information about novel genes that are potential candidate genes in EB. Project 3, "Mutation Analysis in EB: Genotype/Phenotype Correlations and Revised Classification," will provide precise information on the specific mutations in the gene/protein systems that are at fault in various forms of EB. Examination of the mutation database will allow establishment of genotype/phenotype correlations with a profound impact on genetic counseling of the affected individual and families at risk for recurrence of EB. Extended mutation analysis will also form a basis for revised classification of EB subtypes. Project 4, "Functional Consequences of the Mutations at the Protein Level" will examine the expression and secretory pathways of altered proteins in cells cultured from affected individuals. This project will also identify domains critical for protein-protein interactions. Project 5. "Development of Gene Therapy for EB", will concentrate on testing gene therapy approaches towards treatment of EB. This proposal will highlight a novel, highly promising approach utilizing RNA/DNA chimeric oligonucleotides for homologous recombination. This multidisciplinary studies are expected to provide precise information of critical importance for translational applications of research towards development of definitive classification and prenatal testing of EB, as well as providing the basis for novel gene therapy approaches for this devastating group of skin diseases.